Drugs are often metabolized to agents which themselves can elicit therapeutic or toxic responses. Although metabolites can be chemically inert and cause their effects by interacting reversibly with enzymatic receptors, in some cases the metabolites are chemically reactive. These reactive species can covalently bind to tissue macromolecules or catalyze the formation of such toxins as H2O2. In our laboratory nitro drug reduction by flavoenzymes has recently been discovered to proceed through nitro drug anion free radical intermediates, RNO2. Similarly the azo drugs may also be reduced through an anion free radical. The enzymatic formation of these reactive free radical drug metabolites is expected to lead to drug toxicities. The research objectives of the proposed work here are to: a) Extend our investigations of the kinetics and mechanism of nitro anion free radical formation by nitroreductases. b) Search for electron spin resonance spectral evidence of anion free radical metabolites of azo drugs and food dyes. c) Investigate the catalytic formation of superoxide free radical by nitro- and azoaromatic drugs, which is completely analogous to the generally accepted mechanism of paraquat toxicity. The reversal of nitro drug stimulated oxygen uptake by superoxide dismutase has been found to be a useful superoxide assay. d) Determine if the reductive intermediate of nitroheterocyclic drugs that covalently bind to protein is the anion radical. The rate of binding of radioactive drug will be compared to the rate of free radical decay as determined in a parallel electron spin resonance experiment. These studies will attempt to rationalize the toxicities of nitro and azo drugs in terms of the reactions of anion free radical metabolites.